The overall aim of this project is to use multidimensional heteronuclear magnetic resonance (NMR) spectroscopy to characterize the three dimensional (3D) structures, membrane binding mechanisms, multimerization interfaces, and dynamics of biologically important proteins involved in human disease. Each domain is predicted to represent a novel structural fold and has been shown to bind directly to or fold on membranes. The specific targets of our study are: alpha-Synuclein: The alpha-synuclein protein is a dynamic 140 residue protein that folds into a helical structure on acidic membranes and oligomerizes into beta sheet fibrils. The protein is driven into the latter state by mutations linked to Parkinson's disease. The solution structures of the micelle-bound and free states of alpha-synuclein will be elucidated and compared. The structure of the pockets that ligate acidic phospholipids and metals will be defined, as will the sites that become phosphorylated, nitrosylated, or truncated during the course of polymerization. The changes in conformation and dynamics that occur upon oligomerization and folding onto micelle surfaces will be investigated. Glycine Receptor: The glycine receptor is a prototypic member of a superfamily of ion channels gated by glycine, GABA. serotonin, and nicotinic acetylcholine neurotransmitters. We have identified the extracellular domains of the glycine receptor by limited proteolysis and have demonstrated their functional integrity. The 3D structure of the approximately 100 residue neurotransmitter binding domain of this receptor will be characterized by NMR. The structure of the binding sites for agonists, strychnine, phospholipids, zinc, and peptides that signal to the ion channel will be defined. BEACH Domain: The Chediak-Higashi syndrome is a potentially fatal human genetic disorder caused by mutations in the CHSI protein and its BEACH domain that disrupt lysosomal trafficking. We have defined the boundaries of this novel approximately 150 residue domain and have shown that it associates with lipids. Here we elucidate its oligomeric state and 3D structure in the presence of micelles to reveal the structural basis of membrane interaction and protein recognition. DIX Domain: The DIX domain is a novel approximately 85 residue signaling module that plays a key role in the Wnt signaling pathway that contributes to embryonic development and cancer progression. The solution structures of Disheveled and Axin DIX domain homodimers and monomers will be elucidated. The phospholipid and protein binding properties of these domains will be characterized by circular dichroism and fluorescence spectroscopy. The interfaces that mediate heterodimerization and membrane association will be identified by NMR, sedimentation equilibrium, and mutagenesis experiments in order to define the unique signaling roles of these two domains.